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Subcloning site directed Mutants

R. John Lye rjl6n at virginia.edu
Wed Jan 31 09:20:16 EST 2001

jayni76 at my-deja.com wrote:

> I create a lot of point mutants using site directed mutagenesis.  The
> plasmids are usually around 7kb and I use  promega Pfu enzyme.  I have been
> subcloning the mutants after PCR to eliminate other mutations but have been
> informed that people generally no longer bother as the enzymes are efficient.

While it is true that some of the proofreading enzymes are much less
error prone than taq, in my opinion, the more relevant improvement
is in the sequencing. It is now so easy and cheap to verify your mutants
by sequencing them that that has become the prefered method.  At least,
that's what I do.


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